Hyperphosphorylated tau aggregation-based Alzheimer?s disease early drug discovery Alzheimer?s disease (AD) is an irreversible neurodegenerative disease affecting 47 million people worldwide, and costs 604 billion US dollars every year for medical expenses. To date, there is no cure or prevention for AD. The two defining features of AD are A? plaques and neurofibrillary tangles that are composed of hyperphosphorylated tau. While the AD drug discovery landscape has been dominated by anti-A? measures, recurring failures of clinical trials argue strongly that a realignment of the drug target and strategies is needed to make a breakthrough in AD therapeutics development. Indeed, multiple lines of evidence suggest that the pre- tangle stage of hyperphosphorylated tau aggregates cause diffusible cytotoxicity that likely underlies neurodegeneration. Screening for compounds that prevent hyperphosphorylated tau from forming the cytotoxic aggregates thus affords a more viable route for AD drug discovery. The tangle-centric drug design has not come to fruition. A wide spectrum of compounds have been identified in multiple screens as inhibitors of tangle formation, but later found to be false-positive. One shared issue for these screens is the use of an unmodified tau protein that requires an inducer, e.g., heparin, for efficient aggregation in a reducing environment. The assay subject (tau) lacks the pathological mark of hyperphosphorylation, and the disease relevance of the inducer has not been substantiated. To overcome these hurdles, we have developed the PIMAX technology that produces hyperphosphorylated tau (p-tau) in E. coli. Purified p-tau fibrillizes autonomously (without an inducer), and causes apoptosis of different cells including a neuroblastoma cell line. This inducer-free, p-tau aggregation assay has a Z? value of 0.699, and a coefficient of variation (CV) of 8.3%. These parameters qualify our p-tau aggregation assay as a robust HTS platform. Using this assay, we conducted a pilot screen for 1,280 compounds for their ability to modulate the aggregation of p- tau. We then used novel biochemical and cell-based secondary assays to verify the candidate compounds. We found that an active neurological drug is a potent p-tau aggregation inhibitor, which also protects cells from p- tau cytotoxicity. These preliminary studies afford solid evidence for the values of p-tau in the quest for AD therapeutics. This R01 project is the very first that uses the pathophysiologically relevant hyperphosphorylated tau for AD drug discovery. We will follow our pilot screen protocols to conduct a high-throughput screen of 100,000 compounds for their ability to inhibit p-tau aggregation and to protect cells. This early discovery project will conclude with two major products: (1) A collection of chemical hits with confirmed p-tau aggregation inhibitory and cytoprotective activities, and (2) a follow-up proposal with comprehensive plans to identify the optimal chemical lead for AD drug development.